Transforming growth factor-alpha (TGFalpha) is a member of the EGF receptor (EGF-R) ligand family that also includes EGF, amphiregulin, HB-EGF, betacellulin, and epiregulin. Besides being potent mitogens, these proteins regulate the migration and differentiation of various cell types, and may have other activities as well. The EGF family has attracted considerable interest, in part because it exemplifies a category of growth factors that is defined by proteolytic release of soluble ligands from the ectodomains of integral membrane precursors. Additionally, the expression of some members (e.g., TGFalpha, amphiregulin, and HB-EGF) is frequently deregulated in neoplastic cells and tissues, and evidence indicates that this could be an important event in muItistep tumorigenesis. Although the activities of the EGF family members have been well-characterized in cell culture and in various assay systems, their physiological roles have not been defined. Additionally, it is not known whether the membrane-anchored forms have distinct signaling roles, or whether their proteolytic processing, which is poorly characterized, is a regulated event. Finally, the molecular mechanisms that drive induced expression of EGF family ligands in neoplastic cells have not been adequately described. With these issues in mind, the specific aims of this proposal are to: (1) identify functions of EGF family members using a gene targeting approach; (2) determine to what extent, and in which contexts, the activities of the different EGF family members overlap or interact by deriving and characterizing mice containing multiple gene knockouts; (3) test the hypothesis that the integral membrane precursors have distinct physiological roles by creating and characterizing lines of mice that express either soluble growth factors or cleavage-resistant precursors only; and (4) investigate the link between cellular transformation and TGFalpha by (a) identifying mechanisms responsible for activation of TGFalpha gene expression in neoplastic cells, and (b)determining if the proTGFalpha processing enzyme is coinduced in transformation.